Capillary Electrophoresis Device

ABSTRACT

The purpose of this invention has to do with being able to eliminate, using a small amount of an electrophoresis medium, air bubbles that get mixed in when loading an electrophoresis-medium container into a capillary electrophoresis device. This invention has to do with being able to simplify a positive-electrode-side channel in a capillary electrophoresis device by electrophoresing using only an electrophoresis medium on the positive-electrode side. This invention makes it possible to eliminate, easily and using a small amount of an electrophoresis medium, air bubbles that had become mixed in each time an electrophoresis-medium container was connected to the device. This invention also makes it easier to manage consumables and reduces the number thereof, making pre-electrophoresis preparation simple, and makes it possible to simplify and reduce the size of the device.

TECHNICAL FIELD

The present invention relates to a technique for separating andanalyzing the nucleic acid, protein, or the like throughelectrophoresis, and particularly to a capillary electrophoresis device.

BACKGROUND ART

In recent years, a capillary electrophoresis device having a capillaryfilled with a phoresis medium such as polymer gel or polymer solutionhas been widely used.

For example, the capillary electrophoresis device as disclosed in PTL 1has conventionally been used. This device has features of having thehigher heat dissipation property than the flat-type electrophoresisdevice and being capable of faster electrophoresis because the highervoltage can be applied to the sample. Other features are: the necessaryamount of sample is small, filling with the separation medium can beautomatically carried out, the sample injection can also beautomatically carried out, and the like. Such a device is used invarious separation and analysis measurements including the analysis ofthe nucleic acid and protein.

FIG. 1 illustrates an example of the conventional capillaryelectrophoresis device. The capillary electrophoresis device includes acapillary 101, a power source 102 that applies high voltage to both endsof the capillary 101, an illumination system, which is not shown,including a laser light source and the like, a light-reception opticalsystem, which is not shown, for detecting fluorescence, a thermostattank 103 that controls the temperature of the capillary, a phoresismedium filling unit 104 that fills the capillary 101 with the phoresismedium, and a carrier, which is not shown, that carries a containercontaining the sample.

An anode side of the capillary 101 is bonded to a flow channel of thephoresis medium filling unit 104. The flow channel of the phoresismedium filling unit 104 is branched into two channels. One of thechannels is bonded to a phoresis medium container 105 while the other isbonded to a buffer solution container A 106.

In the capillary electrophoresis device, the capillary 101 having aninner diameter of as small as 50 μm needs to be filled with the phoresismedium whose viscosity is several hundred times as high as that ofwater. In view of this, the phoresis medium filling unit 104 has amechanism that can apply the pressure of several megapascals to one endof the channel for the phoresis medium. One example of this type ofmechanism is a plunger pump 107. In the case of FIG. 1, the plunger pump107 is driven in a direction perpendicular to the paper surface. Thedriving of the plunger pump 107 changes the capacity of the channel toproduce the pressure necessary for filling with the phoresis medium.

In the analysis of the sample, high voltage is applied between oppositeends of the flow channel connected to the capillary 101 (between thebuffer solution container A 106 and a buffer solution container B 109),thereby having a fluorescence-labelled sample such as DNA subjected tothe electrophoresis in the phoresis medium of the capillary. Here, thecharges used in the electrophoresis are mostly the charges in the buffersolution on the anode side. The sample differs in the phoresis speeddepending on the molecular size and is detected in the detection unit108.

Incidentally, the capillary electrophoresis device needs the exchange ofthe phoresis medium container 105 or the capillary 101. In the exchangeof these components, part of the flow channel is exposed to the air, inwhich case the air may be mixed into the flow channel.

In the electrophoresis, voltage as high as several to several tens ofkilovolts is applied between the opposite ends of the flow channel.Therefore, if there is an air bubble in the channel, the bubble mayblock the channel electrically. If the channel is electrically blocked,the high voltage difference is caused in the blocked portion, whichresults in the discharge. Depending on the magnitude of the discharge,the capillary electrophoresis device may be destroyed.

In view of the above, it is necessary to remove the air bubble out ofthe flow channel before the start of the electrophoresis.

For example, if there is an air bubble in the flow channel of thephoresis medium filling unit 104, the connected flow channel between thephoresis medium filling unit 104 and the capillary 101 is closed and inthis state, the phoresis medium is supplied to the buffer solutioncontainer A 106 in a manner that the medium returns at the branched pathin the unit. Thus, the air bubble is removed from the flow channelsection of the phoresis medium filling unit 104.

On the other hand, if there is an air bubble in the flow channel of thecapillary 101, the capillary 101 is filled with the phoresis mediumwhose amount is 1.5 times as large as the capacity of the capillary 101.Here, the inner diameter of the capillary 101 is as small as 50 μm.Thus, the air bubble flows inside the capillary 101 together with thephoresis medium and is discharged from the other end of the capillary101. In other words, the air bubble can be removed from the inside ofthe capillary.

PTL 2 discloses the mechanism for removing the air bubble from the flowchannel of the phoresis medium filling unit 104 with a small amount ofphoresis medium. Specifically, the structure is employed which forms theconnected flow channel so that the phoresis medium flows from the bottomto the top in the connected portion between the phoresis medium fillingunit 104 and the capillary 101.

CITATION LIST Patent Literatures

PTL 1: Japanese Patent No. 2776208

PTL 2: JP-A-2008-8621

SUMMARY OF INVENTION Technical Problem

In the case of the conventional device, since the phoresis mediumfilling unit 104 has the long flow channel, a large amount of phoresismedium is consumed in removing the air bubbles from the flow channel.

In view of the above, an object of the present invention is to provide acapillary electrophoresis device with the phoresis medium filling unit104 having the shorter flow channel so as to consume less phoresismedium in removing the air bubbles.

Solution to Problem

In order to achieve the object, in the present invention, theelectrophoresis is carried out with the charges necessary for theelectrophoresis not from the buffer solution but from the phoresismedium, i.e., only with the electrophoresis medium in regard to thecapillary anode end.

Advantageous Effects of Invention

According to the present invention, the flow channel from the capillaryconnected portion to the container containing the buffer solution in thephoresis medium filling unit 104 can be omitted from the flow channel inthe electrophoresis. This can suppress the consumption of the phoresismedium required for removing the air bubble out of the phoresis mediumfilling unit 104.

Furthermore, the buffer solution container 106 is no longer necessary,so that the number of consumption articles can be reduced, which cansimplify the preparation before the analysis and the device. As aresult, it becomes easier to operate the electrophoresis device.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating a conventional example of a capillaryelectrophoresis device.

FIG. 2 is a diagram schematically illustrating an entire structure of anelectrophoresis device according to Example 1.

FIG. 3 is an external diagram of a capillary array.

FIG. 4A is an external structure diagram of a phoresis medium container.

FIG. 4B is a sectional diagram of the phoresis medium container.

FIG. 4C is an external exploded structure diagram of the phoresis mediumcontainer.

FIG. 4D is a structure diagram of a component (lid) of the phoresismedium container.

FIG. 4E is a structure diagram of a component (middle lid) of thephoresis medium container.

FIG. 4F is a structure diagram of a component (rubber film) of thephoresis medium container.

FIG. 4G is a structure diagram of a component (main body portion) of thephoresis medium container.

FIG. 5 is a diagram illustrating a structure of a resin flow channelblock with the high electric insulating property used in Example 1.

FIG. 6 is a diagram illustrating a process step of filling the capillarywith the phoresis medium.

FIG. 7A is a diagram illustrating the flow channel in the resin flowchannel block with the high electric insulating property according to amodified example.

FIG. 7B is a structure diagram in which a hollow pipe is used as anelectrode according to a modified example.

DESCRIPTION OF EMBODIMENTS

An embodiment of the present invention will be hereinafter describedwith reference to the drawings. Note that the device structure and thecontent of the process to be described below correspond to an example ofthe present invention and will not limit the content of the presentinvention. Embodiments can be combined with each other, or theembodiment can be combined with a known technique or replaced by a knowntechnique to achieve another embodiment.

A specific example of the device structure of the electrophoresis devicesuggested by the present inventor is hereinafter described.

Example 1 Summary of System

FIG. 2 schematically illustrates the entire structure of anelectrophoresis device according to Example 1. The electrophoresisdevice according to Example 1 includes a capillary array 202, which is asingle capillary 201 or a group of capillaries 201, a laser light source203 that irradiates a fluorescence-labelled sample in the capillary witha laser beam, a light-reception optical system 204 that detects thefluorescence emitted from the sample, a high-voltage application unit205 that applies high voltage to the capillary, and a thermostat tank206 that maintains the capillary at a constant temperature.

The capillary array 202 is fixed to the thermostat tank 206. Outside thethermostat tank 206 is provided a detection unit 207 that is used fortesting the sample. In the drawing, the side provided with a buffersolution container 208 corresponds to the cathode end of the capillaryarray 202 and also to a sample suction end 209 through which the sampleis injected.

The sample suction end 209 is immersed in a buffer solution 210 in thebuffer solution container 208 while the other (capillary head 302) isconnected to a resin flow channel block 211 with the high electricinsulating property. The resin flow channel block 211 is bonded to ahollow pipe 212 in addition to being bonded to the capillary array 202.This hollow pipe 212 is connected to a phoresis medium container 214containing a phoresis medium 213. In the resin flow channel block 211,an electrode 215 is also installed.

(Structure of Capillary Array)

FIG. 3 is an external diagram of the capillary array 202. Description ishereinafter made with reference to FIG. 2 and FIG. 3. Each capillary 201included in the capillary array 202 has an outer diameter of 0.1 to 0.7mm and an inner diameter of 0.02 to 0.5 mm, and is coated with polyimideresin on the outside. The capillary 201 itself is a quartz pipe, and onecapillary 201 or a plurality of (eight in this example) capillaries 202is arranged to constitute the capillary array 202. The capillary array202 includes a load header 302 that takes the sample into the capillary201 from the sample container containing a fluorescence-labelled DNAsample or the like by the electric operation, the detection unit 207that arranges and fixes the capillaries 201 in the order of the samplenumber of the load header 302, and a capillary head 301 binding andbonding the plural capillaries 201. The sample suction end 209projecting from the load header 302 is provided with a hollow electrodeA 303 for applying the high voltage to the capillary 201. The detectionunit 301 includes an opening 304 through which the aligned and heldcapillary array 202 is irradiated with the laser beam from the side, andan opening 305 through which the light emitted from the capillary isextracted.

In regard to the shape of the connected portion between the capillaryhead 301 of the capillary array 202 and the resin flow channel block211, a sleeve is attached to the round capillary head 301 binding thecapillaries 201, and the sleeve is deformed by fastening a setscrew,thereby filling the space. This enables the capillary head 301 to befixed to the resin flow channel block 211.

(Structure of Phoresis Medium Container)

FIGS. 4(A) to 4(G) illustrate the detailed structure of the phoresismedium container 214 used in the examples. FIG. 4(A) is an externalstructure diagram of the phoresis medium container 214, FIG. 4(B) is asectional structure diagram, FIG. 4(C) is an external exploded structurediagram, and FIG. 4(D) to FIG. 4(G) are external structure diagrams ofthe components.

The phoresis medium container 214 includes a lid 401, a middle lid 402,a rubber film 403, a main body portion 404, and a plunger 405. Therubber film 403 is fixed to the main body portion 404 with the middlelid 402 interposed therebetween when the lid 401 is rotated by a screwportion 406 provided for the lid 401. On this occasion, the middle lid402 is set so that a tapered portion A 407 of the rubber film 403 is nottwisted by the rotation of the lid 401. In this structure, asillustrated in FIG. 00, a protrusion 409 of the middle lid 402 is fittedto a groove 408 of the main body portion 404, and when the lid 401 isfastened, the middle lid 402 transmits only the force in the verticaldirection to the rubber film 403. Further, the hollow pipe 212 ispenetrated through a depressed portion 410 above the rubber film 403.When the phoresis medium 214 is supplied by the plunger 405, the taperedportion A 407 of the rubber film 403 is pressed by a tapered portion B411 of the middle lid 402, whereby the leakage from around the hollowpipe 212 is prevented during the penetration of the hollow pipe 212.

(Structure of Resin Flow Channel Block 211)

FIG. 5 illustrates the structure of the resin flow channel block 211used in Example 1. The resin flow channel block 211 includes the hollowpipe 212 and the electrode 215.

Moreover, the flow channel in the resin flow channel block 211 has thesmaller diameter than the air bubble generated in the flow channel sothat when the capillary 201 is filled with the phoresis medium 213, theair bubble in the flow channel in the resin flow channel block 211 canmove for sure. In this example, the flow channel has an inner diameterof φ0.5 mm.

(Operation of Entire Device)

Next, description is made of a series of process operations of thecapillary electrophoresis device according to this example. Theoperation including the application of voltage for the electrophoresisin the capillary electrophoresis device to be described below isperformed through a control unit (such as a computer), which is notshown.

FIG. 6 illustrates a process step of filling the capillary array 202with the phoresis medium 213.

First, the hollow pipe 212 is penetrated into the phoresis mediumcontainer 214. After that, the plunger 405 of the phoresis mediumcontainer 214 is pressed to inject the phoresis medium 213 into thecapillary 201. On this occasion, the air bubbles mixed into the resinflow channel block 211 and the hollow pipe 212 go through the resin flowchannel block 211 and moreover through the capillary 201 together withthe phoresis medium 213 because the inner diameter of the capillary 201is small, and then is discharged out of the sample suction end 209. Theamount of phoresis medium 213 injected into the capillary 201 is about1.5 times as large as the inner capacity of the hollow pipe 212 and therein flow channel block 211+the inner capacity of the capillary array202. In the flow channel of the resin flow channel block 211 and thephoresis medium container 214, the phoresis medium 213 with the chargesnecessary for one electrophoresis is left. In this example, thecapillary array 202 has a length of 26 cm, 8 channels, and an innerdiameter of φ50 μm. The amount of charges necessary for theelectrophoresis is set to 87 mC from the experiments, and this amount issatisfied by approximately 60 μl of phoresis medium (POP-7™)manufactured by Life Technologies. When the phoresis medium 213 isfilled, the sample suction end 209 is immersed in a waste tank (filledwith pure water), which is not shown, carried by a carrier tray, whichis not shown.

After that, the sample suction end 209 is sank into the samplecontainer, which is not shown, carried by the carrier tray, which is notshown, and then sank into the container containing pure water (forcleaning), which is not shown, and into the buffer solution container208 in this order. After that, the electrophoresis is started in thestate that the sample suction end 209 of the capillary array 202 isimmersed in the buffer solution container 208.

As described above, the use of the electrophoresis device according tothis example can easily remove the air bubbles, which are mixed in thesetting of the phoresis medium container 214 and the capillary array202, with a small amount of phoresis medium 213 and can drasticallyreduce the running cost. Furthermore, the preparation for theelectrophoresis can be facilitated as compared to the conventionaldevice.

Example 2

In the description above, the flow channel of the resin flow channelblock 211 has the circular shape with the diameter smaller than that ofthe air bubble generated in the flow channel, so that the air bubblemoves certainly and is not left in the flow channel. Even if the airbubble is mixed in the resin flow channel block 211, a problem does notoccur as long as the air bubble does not block the flow channel, i.e.,the air bubble is not left in the place where the electrophoresis isinterrupted. For example, the micro-channel may be provided for trappingthe air bubble, which is well known as the flow channel for themicro-chemical chip like the flow channel illustrated in FIG. 7A. In themicro-channel, the air bubble is easily formed on the smaller channelside due to the surface tension. Using this phenomenon, the air bubblemixed in the resin flow channel block 211 is moved toward themicro-channel, so that the wider flow channel can secure the bypassflow. Accordingly, the electrophoresis is not interrupted.

In the above description, the resin flow channel block 211 includes thehollow pipe 212 and the electrode 215. However, the hollow pipe may beused as the electrode and the electrode may be omitted as illustrated inFIG. 7B.

In the above description, the resin flow channel block 211 and thecapillary head 301 are structured as separate parts. However, theseparts may be an integrated component.

REFERENCE SIGNS LIST

-   101 capillary-   102 power source-   103 thermostat tank-   104 phoresis medium filling unit-   105 phoresis medium container-   106 buffer solution container A-   107 plunger pump-   108 detection unit-   109 buffer solution container B-   201 capillary-   202 capillary array-   203 laser light source-   204 light-reception optical system-   205 high-voltage application unit-   206 thermostat tank-   207 detection unit-   208 buffer solution container-   209 sample suction end-   210 buffer solution-   211 resin flow channel block-   212 hollow pipe-   213 phoresis medium-   214 phoresis medium container-   215 electrode-   301 capillary head-   302 load header-   303 hollow electrode A-   304 opening for delivering laser beam-   305 opening for extracting emitted light-   401 lid-   402 middle lid-   403 rubber film-   404 main body portion-   405 plunger-   406 screw portion-   407 tapered portion A-   408 groove-   409 protrusion-   410 depressed portion-   411 tapered portion B

1. A capillary electrophoresis device comprising: a capillary; a buffersolution container where a sample suction end corresponding to one endof the capillary through which a sample is injected is immersed in abuffer solution; a flow channel to which a capillary head correspondingto the other end of the capillary is connected; a phoresis mediumcontainer containing a phoresis medium, which is connected to the flowchannel; and a mechanism for injecting the phoresis medium in thephoresis medium container to the capillary through the flow channel;wherein the phoresis medium container includes a lid, a middle lid, arubber film, a main body portion, and a plunger, the lid is providedwith a screw portion for fixing the lid to the main body portion byrotating the lid, and the main body portion and the middle lid are fixedby being fitted to each other and only a force in a vertical directionis transmitted to the rubber film when the lid is fastened.
 2. Thecapillary electrophoresis device according to claim 1, wherein the flowchannel includes a flow channel block to which the capillary head isconnected, and a hollow pipe connected to the flow channel block.
 3. Thecapillary electrophoresis device according to claim 1, wherein themechanism is a plunger connected to the phoresis medium container and isconfigured to inject the phoresis medium in the phoresis mediumcontainer into the capillary by moving the plunger.
 4. The capillaryelectrophoresis device according to claim 1, further comprising: a laserlight source for delivering a laser beam to a fluorescence-labelledsample in the capillary; a light-reception optical system for detectingfluorescence emitted from the sample; and a high-voltage applicationunit for applying high voltage to the capillary.
 5. The capillaryelectrophoresis device according to claim 1, wherein the high-voltageapplication unit is configured such that an electrode on a cathode sideis disposed in the buffer solution container, an electrode on an anodeside is disposed in the flow channel, and high voltage is applied to thecapillary.
 6. The capillary electrophoresis device according to claim 1,wherein a DNA fragment subjected to the electrophoresis toward the anodeside is pushed toward the cathode side through the capillary.
 7. Thecapillary electrophoresis device according to claim 1, wherein thephoresis medium used in the electrophoresis is discharged toward thecathode side.
 8. The capillary electrophoresis device according to claim1, wherein an electrophoresis path is secured by a micro-channel even ifan air bubble is mixed in a flow channel of a flow channel block.
 9. Thecapillary electrophoresis device according to claim 1, wherein thecapillary head and the flow channel block are integrated.
 10. Thecapillary electrophoresis device according to claim 1, wherein the mainbody portion is provided with a groove and the middle lid is providedwith a protrusion to be fitted into the groove.
 11. The capillaryelectrophoresis device according to claim 1, wherein the rubber film andthe middle lid are provided with a tapered portion, and the taperedportion of the middle lid is pressed against the tapered portion of therubber film when the lid is fixed to the main body portion.